First Report of Aster Yellows Phytoplasma in Grapevines in South Africa.

For many years phytoplasma diseases have caused serious losses in most of the major grape-growing regions of the world, except South Africa, where a mixed phytoplasma infection was first reported in 2006 (1). During the early growing season of 2006, symptoms consistent with phytoplasma disease were observed in vineyards in the Olifants River Valley. Symptoms included yellowing of leaves, incomplete lignification of shoots, shortening of internodes, and the abortion of growth tips and immature bunches. Symptomatic shoots and leaves from grapevine cultivars (Merlot, Shiraz, Cabernet Sauvignon, Ruby Cabernet, Pinotage, Corinth, Chardonnay, Columbar, Chenin blanc, Sauvignon blanc, Sultana, and Regal) were collected during the early growing season (November) of 2006, 2007, and 2008. Total DNA was extracted from 32 of these samples (from single plants in the same vineyards over the 3 years) with the Invisorb Spin Plant Mini Kit (Invitek, Berlin, Germany) and tested by nested PCR using two universal primer pairs, P1/P7 and R16F2n/R16R2 (3). The first round of PCR of the 2006 samples yielded 1.8-kb fragments for 17 of the samples, while the nested PCR yielded an additional seven positive samples, confirming the necessity of nested PCR for reliable diagnosis. A similar trend was observed in the 2007 and 2008 PCR test results. All asymptomatic plants, which were included as negative controls, and water controls were negative by nested PCR. Twenty-four 1,245-bp amplicons, generated by nested PCR, were excised from gels, purified with a NucleoSpin Extract II Kit (Macherey-Nagel, Düren, Germany) and directly sequenced. Sequence data was compiled with the BioEdit Version 7.0.4.1 sequence alignment editor software (2), aligned using ClustalW Version 1.4 (4), and a consensus sequence was generated (GenBank Accession No. GQ365729). A BLAST search of the NCBI GenBank database using the individual sequences revealed high sequence identities (≥99%) with the aster yellows phytoplasma group (16SrI) and specifically with the subgroup 16SrI-B. In a comparison of the sequences of the 1.2-kb PCR fragments of 24 local samples with each other, sequence identities of ~99% were observed. These results clearly illustrate that all vines screened were infected with the same phytoplasma. Single nucleotide differences observed between different isolates may indicate the presence of closely related sequence variants of this phytoplasma. Aster yellows occurs worldwide and has been reported to infect grapevine-South Africa can now be added to this list. During the three seasons of our study, the area in which symptomatic vineyards were observed increased significantly, indicating spread by a biological vector. Moreover, infected vineyards were noticed in two other South African grape-growing regions. In contrast to the previous report, which reported a mixed infection of phytoplasmas of groups 16SrXII-A and 16SrII-B (1), PCR screening and sequencing of more than 40 individual samples from these areas confirmed these all to be infected with aster yellows phytoplasma only. To our knowledge, this is the first report of the detection and identification of an aster yellows phytoplasma causing grapevine yellows disease in South Africa. References: (1) S. Botti and A. Bertaccini. Plant Dis. 90:1360, 2006. (2) T. A. Hall. Nucleic Acids. Symp. Ser. 41:95, 1999. (3) I.-M. Lee et al. Phytopathology 83:834, 1993. (4) J. D. Thompson et al. Nucleic Acids Res. 22:4673, 1994.